Antithrombotic compounds

ABSTRACT

The present invention relates to compounds of formula (1), wherein R 1  is phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, (iso)quinolinyl, tetrahydro(iso)quinolinyl, 3,4-dihydro-1H-isoquinolinyl, chromanyl or the camphor group, which groups may optionally be substituted with one or more substitutents selected from (1-8C)alkyl or (1-8C)alkoxy; R 2  and R 3  are independently H or (1-8C)alkyl; R 4  is (1-8C)alkyl or (3-8C)cycloalkyl; or R 3  and R 4  together with the nitrogen atom to which they are bonded are a nonaromatic (4-8)membered ring optionally containing another heteroatom, the ring optionally being substituted with (1-8C)alkyl or SO 2 -(1-8C)alkyl; Q is a spacer having a chain length of 10 to 70 atoms; and Z is a negatively charged oligosaccharide residue comprising two to six monosaccharide units, the charge being compensated by positively charged counterions; or a pharmaceutically acceptable salt thereof or a prodrug thereof. The compounds of the invention have antithrombotic activity an can be used in treating or preventing thrombin-related diseases.

The invention relates to new antithrombotic agents, a process for theirpreparation, pharmaceutical compositions containing the compounds asactive ingredients, as well as the use of said compounds for themanufacture of medicaments.

Serine proteases are enzymes which play an important role in the bloodcoagulation cascade. Members of this group of proteases are for examplethrombin, trypsin, factors VIIa, IXa, Xa, XIa, XIIa, and protein C.Thrombin is the final serine protease enzyme in the coagulation cascade.The prime function of thrombin is the cleavage of fibrinogen to generatefibrin monomers, which are cross-linked to form an insoluble gel. Inaddition, thrombin regulates its own production by activation of factorsV and VIII earlier in the cascade. It also has important actions atcellular level, where it acts on specific receptors to cause plateletaggregation, endothelial cell activation and fibroblast proliferation.Thus thrombin has a central regulatory role in haemostasis and thrombusformation. Since inhibitors of thrombin may have a wide range oftherapeutical applications, extensive research is done in this area.Another important serine protease, factor Xa, catalyzes the conversionof prothrombin into thrombin.

In the development of synthetic inhibitors of serine proteases, and morespecifically of thrombin, the benzamidine moiety is one of the keystructures. It mimics the protonated side-chain of the basic amino acidsArg and Lys of its natural substrates. Compounds with this moiety havebeen studied extensively and repeatedly. A very potent representative ofthis type of thrombin inhibitors is the amino acid derivativeNα-(2-naphthylsulfonyl)-glycyl-4-amidinophenylaianinpiperidide (NAPAP)(Stürzebecher, J. et al., Thromb. Res. 29, 635-642, 1983). However, theprofile of NAPAP is unattractive for therapeutical applications: forexample, the compound has low thrombin specificity and is poorlysoluble. Derivatives of NAPAP were subsequently prepared, such as theN-alkyl substituted derivatives disclosed in EP 0,236,163 or theglycopeptide derivatives described EP 0,558,961, Proc. Am. Pept. Symp.,13th (60LXAW); 94; pp. 643-5 (Stüber, W. et al., Pept.: Chem., Struct.Biol.,), Proc. Int. Symp. Controlled Release Bioact. Mater. (PCRMEY,10220178); 94;Vol. 21 st; pp. 712-12 (Walter, E. et al.), and EP0,513,543. However, although these derivatizations may have led toimprovements of the pharmacological profile when compared to NAPAP, allsuch NAPAP-derived compounds are still active only as direct thrombininhibitors and they have a restricted plasma half-life and a fastclearance (thus displaying their anti-thrombin activity only for a shortperiod of time).

It has now been found that compounds of the formula (I)

wherein

R¹ is phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl, (iso)quinolinyl,tetrahydro(iso)quinolinyl, 3,4-dihydro-1H-isoquinolinyl, chromanyl orthe camphor group, which groups may optionally be substituted with oneor more substituents selected from (1-8C)alkyl or (1-8C)alkoxy;

R² and R³ are independently H or (1-8C)alkyl;

R⁴ is (1-8C)alkyl or (3-8C)cycloalkyl;

or R³ and R⁴ together with the nitrogen atom to which they are bondedare a nonaromatic (4-8)membered ring optionally containing anotherheteroatom, the ring optionally being substituted with (1-8C)alkyl orSO₂-(1-8C)alkyl;

Q is a spacer having a chain length of 10 to 70 atoms; and

Z is a negatively charged oligosaccharide residue comprising two to sixmionosaccharide units, the charge being compensated by positivelycharged counterions;

or a pharmaceutically acceptable salt thereof or a prodrug thereof arepotent and highly versatile antitbrombotics. The compounds of theinvention have antithrombin activity, but also the structure of thecompounds may be selectively modified so that they have a tuneable mixedprofile of both non-mediated, direct anti-thrombin (factor IIa) activityAnd antithrombin III (AT-III) mediated anti-Xa activity. The compoundsof the invention thus are dual inhibitors. Compounds of the inventionhave a long plasma half-life and, as a result, they possess prolongedanti-thrombin activity compared to NAPAP or its above reportedderivatives. Further, compounds of the invention may escape theneutralizing action of platelet factor 4 (PF4). Low toxicity is also anadvantageous aspect of compounds of this invention.

Another type of dual inhibitors is disclosed in EP 0,649,854. Contraryto the compounds of the present invention the conjugated saccharidecompounds disclosed in that document display indirect, AT-III mediatedanti-thrombin activity, in addition to AT-III mediated anti-Xa activity.

The compounds of the present invention are useful for treating andpreventing thrombin-mediated and thrombin-associated diseases. Thisincludes a number of thrombotic and prothrombotic states in which thecoagulation cascade is activated which include, but are not limited to,deep vein thrombosis, pulmonary embolism, thrombophlebitis, arterialocclusion from thrombosis or embolism, arterial reocclusion during orafter angioplasty or thrombolysis, restenosis following arterial injuryor invasive cardiological procedures, postoperative venous thrombosis orembolism, acute or chronic atherosclerosis, stroke, myocardialinfarction, cancer and metastasis, and neurodegenerative diseases. Thecompounds of the invention may also be used as anticoagulants inextracorporeal blood circuits, as necessary in dialysis and surgery. Thecompounds of the invention may also be used as in vitro anticoagulants.

The mixed profile of the compounds of the invention may be tuned byvarying the nature of the oligosaccharide residue Z and the length ofthe spacer Q. A range of profiles is thereby available.

Any negatively charged oligosaccharide residue of 2 to 6 saccharideunits is useable in the compounds of the present invention. Suitablecompounds of the invention are compounds wherein Z is a sulfated orphosphorylated oligosaccharide residue. Preferably, the oligosaccharideresidue Z is derived from an oligosaccharide which has per se AT-IIImediated anti-Xa activity, such as the saccharides disclosed in EP0,454,220 and EP 0,529,715. Particularly preferred oligosaccharideresidues are pentasaccharide residues. Most preferably, Z has theformula (II)

wherein R⁵ is independently OSO₃ ⁻ or (1-8C)alkoxy.

Further preferred compounds of the invention are compounds of formula I,wherein R¹ is phenyl, 4-methoxy-2,3,6-trimethylphenyl or naphthyl. Inpreferred compounds, NR³R⁴ represents the piperidinyl group. Preferably,R² is H.

The chemical structure of the spacer is of minor or no importance forthe anti-thrombotic activity of the compounds of the invention, it mayhowever not be completely rigid. Highly flexible spacers are moresuitable than others.

Further, for synthetic reasons some spacers are more appropriate thanothers. Suitable spacers that can easily be used have for example theformula (III):

—[(CH₂)₂O]_(m)—[(CH₂)_(n)—NR³—C(O)]_(p)—W—(CH₂)_(s)—  (III),

wherein

W

is —[1,4-phenylene-NR³—C(O)]_(q)—(CH₂)_(r)—S— or

—(CH₂)_(t)—S—(CH₂)_(u)—[O(CH₂)₂]_(v)—O—(CH₂)_(w)—C(O)—NR³—;

and R³ is independently H or (1-8C)alkyl;

m=1-12; n=1-8; p=0-4; q=0 or 1; r=1-8; s=1-8; t=1-8; u=1-8; v=1-12;w=1∝8; the total number of atoms is 10-70; and the moiety—[(CH₂)₂O]_(m)— is the end with which Q is attached to Z.

Preferred spacers are the following:

—[(CH₂)₂O]₅—(CH₂)₂—NH—C(O)—CH₂—S—CH₂—;

—[(CH₂)₂O]₅—(CH₂)₂—NH—C(O)—CH₂—S—(CH₂)₂—[O(CH₂)2]₃—O—CH₂—C(O)—NH—(CH₂)₄—;and

—[(CH₂)₂O]₃—(CH₂)₂—NH—C(O)-1,4-phenylene-NH—C(O)—CH₂—S—CH₂—.

In the description of the compounds of formula (I) the followingdefinitions are used.

The term (1-8C)alkyl means a branched or unbranched alkyl group having1-8 carbon atoms, for example methyl, ethyl, propyl, isopropyl, butyl,sec-butyl, tert-butyl, hexyl and octyl. Methyl and ethyl are preferredalkyl groups.

The term (1-8C)alkoxy means an alkoxy group having 1-8 carbon atoms, thealkyl moiety having the meaning as previously defined. Methoxy is apreferred alkoxy group.

The term (3-8C)cycloalkyl means a cycloalkyl group having 3-8 carbonatoms, being cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl or cyclo-octyl. Cyclopentyl and cyclohexyl are preferredcycloalkyl groups.

The spacer length is the number of atoms of the spacer, counted alongthe shortest chain between Z and the peptide part of the molecule, notcounting the oxygen atom of the oligosaccharide Z which is connected tothe spacer.

The term “prodrug” means a compound of the invention in which the aminogroup of the amidino-moiety is protected, e.g. by hydroxy or a(1-6C)alkoxycarbonyl group.

The compounds of the present invention are prepared by derivatizingNAPAP (or a NAPAP-analogue) at the glycine position with cysteine orlysine using methods generally known in the art, which compoundsubsequently (a) is coupled to a oligosaccharide-spacer residue or (b)is coupled to a spacer, which then is derivatized with a thiol group andsubsequently is coupled to an oligosaccharide residue. Any suitableoligosaccharide may be used for this purpose, for exampleoligosaccharides known in literature (e.g. from EP 0,454,220 and EP0,529,715, but not limited to these sources) or commercially availableoligosaccharides. The oligosaccharides may be phosphorylated at anappropriate time by methods e.g. described by Buijsman, R. et al.(Abstracts of papers, 9th European Carbohydrate Symposium Utrecht 1997,Abstract A150). The coupling of the spacer to the oligosaccharide canfor instance be performed by using the methods described in EP0,649,854.

The peptide coupling, a procedural step in the above described method toprepare the compounds of the invention, can be carried out by methodscommonly known in the art for the coupling—or condensation—of peptidefragments such as by the azide method, mixed anhydride method, activatedester method, or, preferably, by the carbodiimide method, especiallywith the addition of catalytic and racemisation suppressing compoundslike N-hydroxysuccinimide and N-hydroxybenzotriazole. An overview isgiven in The Peptides, Analysis, Synthesis, Biology, Vol 3, E. Gross andJ. Meienhofer, eds. (Academic Press, New York, 1981).

Amine functions present in the compounds may be protected during thesynthetic procedure by an N-protecting group, which means a groupcommonly used in peptide chemistry for the protection of an α-aminogroup, like the tert-butyloxycarbonyl (Boc) group, the benzyloxycarbonyl(Z) group, the 9-fluorenylmethyloxycarbonyl (Fmoc) group or thephthaloyl (Phth) group. Removal of the protecting groups can take placein different ways, depending on the nature of those protecting groups.Usually deprotection takes place under acidic conditions and in thepresence of scavengers. An overview of amino protecting groups andmethods for their removal is given in the above mentioned The Peptides,Analysis, Synthesis, Biology, Vol 3.

The compounds of the invention, which can occur in the form of a freebase, may be isolated from the reaction mixture in the form of apharmaceutically acceptable salt. The pharmaceutically acceptable saltsmay also be obtained by treating the free base of formula (I) with anorganic or inorganic acid such as HCl, HBr, HI, H₂SO₄, H₃PO₄, aceticacid, propionic acid, glycolic acid, maleic acid, malonic acid,methanesulphonic acid, fumaric acid, succinic acid, tartaric acid,citric acid, benzoic acid, ascorbic acid and the like.

The compounds of this invention possess chiral carbon atoms, and maytherefore be obtained as a pure enantiomer, or as a mixture ofenantiomers, or as a mixture containing diastereomers. Methods forobtaining the pure enantiomers are well known in the art, e.g.crystallization of salts which are obtained from optically active acidsand the racemic mixture, or chromatography using chiral columns. Fordiastereomers straight phase or reversed phase columns may be used.

The compounds of the invention may be administered enterally orparenterally. The exact dose and regimen of these compounds andcompositions thereof will neccessarily be dependent upon the needs ofthe individual subject to whom the medicament is being administered, thedegree of affliction or need and the judgment of the medicalpractitioner. In general parenteral administration requires lowerdosages than other methods of administration which are more dependentupon absorption. However, the daily dosages are for humans preferably0.001-100 mg per kg body weight, more preferably 0.01-10 mg per kg bodyweight. The medicament manufactured with the compounds of this inventionmay also be used as adjuvant in acute anticoagulant therapy. In such acase, the medicament is administered with other compounds useful intreating such disease states. Mixed with pharmaceutically suitableauxiliaries, e.g. as described in the standard reference, Gennaro etal., Remington's Pharmaceutical Sciences, (18th ed., Mack PublishingCompany, 1990, see especially Part 8: Pharmaceutical Preparations andTheir Manufacture) the compounds may be compressed into solid dosageunits, such as pills, tablets, or be processed into capsules orsuppositories. By means of pharmaceutically suitable liquids thecompounds can also be applied in the form of a solution, suspension,emulsion, e.g. for use as an injection preparation, or as a spray, e.g.for use as a nasal spray.

For making dosage units, e.g. tablets, the use of conventional additivessuch as fillers, colorants, polymeric binders and the like iscontemplated. In general any pharmaceutically acceptable additive whichdoes not interfere with the function of the active compounds can beused. Suitable carriers with which the compositions can be administeredinclude lactose, starch, cellulose derivatives and the like, or mixturesthereof, used in suitable amounts.

The invention is further illustrated by the following examples.

EXAMPLES

Abbreviations used:

DMAP=N,N-dimethylaminopyridine

TEA=triethylamine

Z=benzyloxycarbonyl

Ac=acetyl

MMTr=monomethoxytrityl

Bn=benzyl

DCHA=dicyclohexylammonium

EDCI=1-(3-dimethylarninopropyl)-3-ethylcarbodiimide hydrochloride

HOBt=1-hydroxybenzotriazole

DiPEA=diisopropylethylamine

Pyr=pyridinyl

TEG=tetraethylene glycol

HEG=hexaethylene glycol

APA=amidinophenylalanine

Cys=cysteine

The numbers of the compounds refer to the compounds on the formulasheets.

4-O-(4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl)-2,3,6-tri-O-acetyl-α-D-glucopyranosyl)-2,3,6-tri-O-acetyl-α/β-D-glucopyranosyltrichloroacetimidate (4)

To a stirred solution of maltotriose (1) (2.0 g, 4.0 mmol) in pyridine(100 mL) was added acetic anhydride (6.2 mL, 65 mmol) and a catalyticamount of DMAP (0.79 g, 6.5 mmol). After 5 h the reaction mixture waspoured into aqueous sodium hydrogencarbonate (1 M, 250 mL) and extractedthree times with ethyl acetate (200 mL). The combined organic layerswere dried on magnesium sulfate and concentrated in vacuo. The productwas purified by column chromatography (light petroleum/ethyl acetate,1/1 to 0/1, v/v) giving 2 as a white foam (91% yield, 3.5 g). Anomericdeacetylation was achieved by treatment of 2 (3.0 g, 3.1 mmol) with 0.1M solution of hydrazine acetate in dimethylformamide (34 mL, 3.4 mmol)for 1 h. After concentration in vacuo the reaction mixture was dilutedwith ethyl acetate (50 mL), washed with sodium hydrogencarbonate (1 M,3×25 mL), dried (magnesium sulfate) and concentrated. Purification bysilica gel column chromatography (light petroleum/ethyl acetate, 3/2 to1/0, v/v) gave 3 (92% yield, 2.7 g). Compound 3 (2.7 g, 3.1 mmol) wasdissolved in dichloromethane (15 mL) and trichloroacetonitrile (1.7 mL)together with a catalytic amount of cesium carbonate (0.2 g, 0.62 mmol)were added. After 1 h the reaction mixture was filtered and the filtratewas concentrated under reduced pressure. Purification of the crude 4 bycolumn chromatography (light petroleum/ethyl acetate/TEA, 50/49/1 to0/99/1, v/v/v) yielded pure 4 as white foam (1.9 g, 71%).

N-Benzyloxycarbonyl-1-aminohexaethylene glycol4-O-(4-O-(2,3,4,6-tetra-O-acetyl-α-D-glucopymanosyl)-2,3,6-tri-O-acetyl-α-D-glucopyraosyl)-2,3,6-tri-O-acetyl-β-D-glucopyranoside(6)

A solution of donor 4 (0.69 g, 0.76 mmol) and acceptor 5 (0.31 g, 0.76mmol) in dichloromethane (1.5 mL) was stirred for 1 h under a flow ofargon in the presence of activated molecular sieves 4 Å (250 mg). Thesolution was cooled to −20° C. and a solution of trimethylsilyltrifluoromethanesulfonate (15 μL) in dichloromethane (0.6 mL) was addeddropwise to the reaction mixture. After 10 min, TLC analysis (5%methanol in dichloromethane) showed the presence of one product. Solidsodium hydrogencarbonate (0.3 g) was added to the reaction mixture,which was stirred for 10 min and then filtrated. The filtrate wasdiluted with dichloromethane (50 mL), subsequently washed with aqueoussodium hydrogencarbonate (1 M, 2×25 mL), dried (magnesium sulfate), andconcentrated in vacuo. The residue was chromatographed on silica gel(0-4% methanol in ethyl acetate) yielding pure 6 (0.57 g, 56% yield).

N-Benzyloxycarbonyl-1-aminohexaethylene glycol4-O-(4-O-(α-D-glucopyranosyl)-α-D-glucopyranosyl)-β-D-glucopyranoside(7)

Compound 6 (0.57 g, 0.43 mmol) was treated with a solution of potassiumtert-butylate (43 mg, 10 mg per mmol Ac) in methanol (15 mL). After 1 hTLC analysis (ethyl acetate/pyrdine/acetic acid/water, 5/7/4/1.6,v/v/v/v) indicate a complete conversion of 6 into 7. The reaction wasneutralized with Dowex 50 WX4-W⁺ resin. The resin was removed byfiltration and the filtrate was concentrated under reduced pressure toafford 7 (0.37 g, 95% yield), which was used without furtherpurification.

N-Benzyloxycarbonyl-1-aminohexaethylene glycol4-O-(4-O-(α-D-glucopyranosyl-2,3,4,6-tetrakis-(dibenzylphosphate))-α-D-glucopyranosyl-2,3,6-tris(dibenzylphosphate))-β-D-glucopyranoside2,3,6-tris(dibenzylphosphate)(9)

A solution of 1H-tetrazole (54 mg, 0.77 mmol) in acetonitril (1 mL) wasaddded to a mixture of 7 (86 mg, 95 μmol) and 8 (450 mg, 1.4 mmol) inacetonitril/dioxane (2/1, v/v, 2 mL). After stirring for 1 h at 20° C.,the reaction mixture was cooled with an ice bath andtert-butylhydroperoxide (0.75 mL) was added. Stirring was continued for45 min, afterwhich TLC analysis showed the presence of one main product.Purification by silica gel column chromatography (100/0 to 95/5,dichloromethane/methanol, v/v) furnished pure 9 (311 mg, 92% yield).

1-Aminohexaethylene glycol 4-O-(4-O-(α-D-glucopyranosyl 2,3,4,6-tetrakisphosphate)-α-D-glucopyranosyl 2,3,6-triphosphate)-β-D-glucopyranoside2,3,6-triphosphate (10)

Compound 9 (311 mg, 87 μmol) was dissolved in tert-butanol/water (6/1,v/v, 20 mL) containing a few drops of acetic acid. The solution wasstirred under a continuous stream of hydrogen in the presence of 10%Pd/C (100 mg). After 3 h the Pd/C catalyst was removed by filtration andthe filtrate was concentrated in vacuo. Dowex 50 WX4Na⁺ ion-exchangethen furnished 10 (179 mg, 98% yield).

N-2-Naphtalenesulfonyl-S-4-monomethoxytrityl-(L)-cysteine (12)

To a stirred mixture of commercially availableS-4-monomethoxytrityl-(L)-cysteine (11) (0.34 g, 1 mmol), dioxane (5 mL)and 10% aqueous sodium carbonate (5 mL) was added 2-naphtalenesulfonylchloride (0.25 g, 1.1 mmol). After stirring for 1 h, the reactionmixture was acidified by addition of 5% aqueous citric acid (50 mL) andextracted with ethyl acetate (2×50 mL). The combined organic layers weredried (magnesium sulfate) and concentrated under reduced pressure. Thecrude product was chromatographed on silica gel(methanol/dichloromethane/triethylamine, 0/99/1 to 4/95/1, v/v/v) toyield 12 (76% yield, 0.44 g).

N-2-Naphtalenesulfonyl-S-2-pyridinesulfenyl-(L)-cysteine (14)

A solution of trifluoroacetic acid and triisopropylsilane indichloromethane (1/1/18, v/v/v) was added to compound 12 (0.44 g, 0.76mmol). After stirring for 20 min, the mixture was poured into water andextracted with dichlorometbane (2×50 mL). The combined organic layerswere dried on magnesium sulfate and concentrated in vacuo. Traces oftrifluoroacetic acid in the crude mixture were removed by coevaporationwith toluene. The resulting free thiol 13 was redissolved in isopropanol(2.5 mL) and added dropwise to a solution of Aldrithiol™ (1.7 g, 7.6mmol) in isopropanol/2 N aqueous acetic acid (1/1, v/v, 20 mL). After 1h, TLC analysis indicated the reaction to be complete and the mixturewas concentrated under reduced pressure. Traces of acetic acid in theresidue were removed by coevaporation with toluene. The crude productwas dissolved in acetone (10 mL) and to this solution dicyclohexylamine(0.3 mL) was added, afterwhich compound 14 precipitated from thereaction mixture as its DCHA-salt. The precipitate was isolated,dissolved in ethyl acetate (50 mL) and washed with 5% aqueous citricacid (2×30 mL). The organic layer was dried (magnesium sulfate) andconcentrated under reduced pressure to afford pure 14 (55% yield, 0.25g).

N(N(N^(α)-2-Naphtalenesulfonyl-S-2-pyridinesulfenyl-(L)-cysteinyl)-(D,L)-4-amidinophenylalanyl)piperidine (16)

To a solution of N-((D,L)-4amidinophenylalanyl)piperidinedihydrochloride (15) (0.13 g, 0.39 mmol) and cysteine derivative 14(0.16 g, 0.39 mmol) in dimethylformamide (2 mL) was added HOBt (58 mg,0.42 mmol), EDCI (82 mg, 0.42 mmol) and N-ethylmorpholine (110 μL, 0.78mmol). After stirring for 16 h, the mixture was diluted withdichloromethane (20 mL) and washed with water (2×10 mL). The organiclayer was dried (magnesium sulfate) and concentrated in vacuo. Theresidue was purified by silica gel column chromatography (first 10%-20%methanol in dicbloromethane to remove impurities and then ethylacetate/pyridine/acetic acid/water (16/7/1.6/4, v/v/v/v) to releaseproduct) and subsequently by gel filtration on Sephadex LH-20 (eluent:methanol/dichloromethane, 4/1, v/v) to yield homogeneous 16 (70%, 0.19g).

Condensation Coupling of Maltotriose-decaphosphate 18 with Peptide 16

To a solution of maltotriose-decaphosphate 18 (21 mg, 9.8 μmol) in 0.1 MNa₂HPO₄ buffer (1.0 mL, pH 7.5) was added a solution ofN-hydroxysuccinimidyl-2-bromoacetate in methanol (1 mL). After stirringfor 2 h, the reaction mixture was applied on a Sephadex G25 columneluted with 10% acetonitril in water. The appropriate fractions werepooled and concentrated under reduced pressure at low temperature (25°C.) to yield compound 19. NAPAP analogue 16 (10 mg, 15 μmol) wasdissolved in a mixture of methanol (1 mL) and 0.1 M Na₂HPO₄ buffer (0.75mL, pH 7.0) degassed by passing through helium and by sonification. Tothis solution tributylphosphine (4.1 μL, 16 μmol) was added and thereaction mixture was stirred under an argon atmosphere. After 1 h HPLCanalysis (Lichrospher® RP18-column) indicated a complete cleavage of the2-pyridinesulfenyl group and a solution of compound 19 indimethylformamide (0.25 mL) and 0.1 M Na₂HPO₄ buffer (0.50 mL, pH 7.0)was added to the reaction mixture. The mixture was stirred for 3 h,afterwhich the crude mixture was purified by gel filtration (FractogelHW-40, eluent: 0.15 M TEAB). Concentration of the appropriate fractionsand subsequent Dowex 50 WX-Na⁺ ion-exchange gave after lyophilizationhomogeneous conjugate I (10.1 mg, 47% yield). The two diastereoisomerswere separated by semi-preparative HPLC column chromatography(LiChrospher® RP-18 column, gradient: 17.5%-22.5% CH₃CN in 0.05 Maqueous TEAA) to give diastereoisomer I-a (retention time: 28.6 min) anddiastereoisomer I-b (retention time: 33.0 min). The two isomers weredesalted by gel filtration (Sephadex G-25 DNA-grade Superfine),transformed into the Na⁺-form using Dowex 50 WX-Na⁺ ion exchange resin.

Diastereoisomer I-a: ¹H NMR (D₂O, 600 MHz, HH-COSY): maltotriose:(reducing end) 4.65 (bs, 1H, H1), 3.85 (m, 1H, H2), 4.35 (m, 1H, H3),3.76 (m, 1H, H4); 5.50 (bs, 1H, H1′), 4.18 (m, 1H, H2′), 4.10 (m, 1H,H4′); (non-reducing end) 5.71 (bs, 1H, H1″), 4.09 (m, 1H, H2″), 4.45 (m,1H, H3″), 4.15 (m, 1H, H4″); 3.95-3.84 (H5, maltotriose); spacer:3.65-3.51 (m, 22H, OCH₂ HEG), 3.35 (m, 2H, CH₂NH₂), 3.15 (s, 2H,SCH₂(O)); peptide: 8.31 (s, 1H, H_(arom) NAS), 8.06-7.67 (m, 6H,H_(arom) NAS), 7.70, 7.17 (2×d, 4H, H_(arom) APA, J=7.8 Hz), 4.28 (m,1H, αCH APA), 3.91 (m, 1H, αCH Cys), 3.30-3.04 (m, 4H, CH₂N piperidine),2.82-2.62 (m, 3H, βCH₂ Cys, βCH APA), 2.57 (m, 1H, βCH′ APA), 1.45-1.25(m, 6H, CH₂ piperidine);

ES-MS: [M−3H]³⁻724.1, [M−2H]²⁻1086.7.

Diastereoisomer I-b: ¹H NMR (D₂O, 600 MHz HH-COSY): maltotriose:(reducing end) 3.80 (m, 1H, H2), 4.32 (m, 1H, H3), 3.89 (m, 1H, H4);5.49 (bs, 1H, H1′), 4.22 (m, 1H, H2′), 4.11 (m, 1H, H4′); (non-reducingend) 5.70 (bs, 1H, H1″), 4.22 (m, 1H, H2″), 4.52 (m, 1H, H3″), 4.24 (m,1H, H4″); 3.91-3.84 (H5, maltotriose); spacer: 3.63-3.52 (m, 22H, OCH₂HEG), 3.35 (t, 2H, CH₂NH₂), 3.17 (AB, 2H, SCH₂(O)); peptide: 8.35 (s,1H, H_(arom) NAS), 8.07-7.65 (m, 6H, H_(arom) NAS), 7.77, 7.22 (2×d, 4H,H_(arom) APA, J=7.8 Hz), 4.62 (t, 1H, αCH APA, J_(αCH,βCH)=7.3 Hz), 4.05(m, 1H, αCH Cys), 3.05-3.00 (m, 4H, CH₂N piperidine), 2.85-2.67 (m, 4H,βCH₂ Cys, βCH₂ APA), 1.88-1.24 (m, 6H, CH₂ piperidine);

ES-MS: [M−3H]³⁻724.0, [M−2H]²⁻1086.2.

N-Hydroxysuccinimidyl-14-S-2-pyridinesulfenyl-14-mercapto-3,6,9,12-tetraoxatetradecanoate(22)

Spacer 20 (0.75, 2.4 mmol) (P. Westerduin et al., Angew. Chem. Int. Ed.Engl. 1996, 35, 3, p331-333) and Aldrithiol™ (2.6 g, 12.1 mmol) wasdissolved in dichloromethane (20 mL) and treated with n-butylamine (4mL). After stirring for 2 h, the reaction mixture was concentrated invacuo, redissolved in dichloromethane (50 mL) and washed with 5% aqueouscitric acid (2×50 mL). The organic layer was dried and concentratedunder reduced pressure. Silica gel column chromatography(methanol/acetic acid/dichloromethane, 0/1/99 to 6/1/93, v/v/v) of theresidue yielded pure 21 (0.80 g, 88% yield). Compound 21 (0.80 g, 2.1mmol) was dissolved in dichloromethane (10 mL) and N-hydroxysuccinimide(0.26 g, 2.3 mmol) and EDCI (0.45 mg, 2.3 mmol) were added to thissolution. After 1 h, the reaction mixture was diluted withdichloromethane (50 mL), washed three times with ice water (20 mL),dried (magnesium sulfate) and concentrated to give 22 (0.98 mg, 98%yield,), which was used without further purification.

N^(ε)-tert-Butyloxycarbonyl-N^(α)-benzenesulfonyl-(L)-lysine (24)

Prepared as described for 12, using 23 and benzenesulfonyl chloride asstarting materials. (0.86 g, 75% yield).

N^(ε)-(14-S-2-Pyridinesulfenyl-14-mercapto-3,6,9,12-tetraoxatetradecanoyl)-N^(α)-benzenesulfonyl-(L)-lysine(26)

Compound 24 (0.86 g, 2.2 mmol) was treated with 3 N hydrogen chloride inethyl acetate. After 15 min the reaction mixture was concentrated invacuo. Traces of acid in the residue were removed by coevaporation withtoluene. The crude 25 was dissolved in a mixture of dioxane/water (4/1,v/v, 2.5 mL) and to this solution compound 22 (0.98 g, 2.1 mmol) andDiPEA (1.1 mL, 6.6 mmol) were added. After 1 h, the reaction mixture wasdiluted with dichloromethane (100 mL) and washed with 5% aqueous citricacid (2×50 mL). The organic layer was dried (magnesium sulfate) andconcentrated in vacuo. The residual oil was purified by silica gelcolumn chromatography (0-10% methanol/ethyl acetate) to give homogeneous26 (0.95 g, 67% yield).

N(N(N^(ε)-(14-S-2-Pyrinesulfenyl-14-mercapto-3,6,9,12-tetraoxatetradecanoyl)-N^(α)-benzenesulfonyl-(L)-lysinyl)-(D,L)-4-amidinophenylalanyl)piperidine(27)

Prepared as described for 16, using 26 and 15 as starting materials. (87mg, 70% yield).

Condensation Coupling of Maltotriose-decaphosphate 18 with Peptide 27(II)

Prepared as described for I, using 18 and 27 as starting materials.Purification of the crude II was effected by semi-preparative HPLC(LiChrospher® RP-18 column). Subsequent desalting by gel filtration(Sephadex G-25 DNA-grade Superfine), transformation into the Na⁺-formusing Dowex 50 WX4-Na⁺ ion exchange resin and lyophilization affordedpure II as a white fluffy solid (8.5 mg, 23% yield from 18).

¹H NMR (D₂O, 600 MHz, HH-COSY): maltotriose: (reducing end) 4.67 (m, 1H,H1), 4.07 (m, 1H, H2), 4.40 (m, 1H, H3), 4.06 (m, 1H, H4); 5.49 (bs, 1H,H1′), 4.24 (m, 1H, H2′), 4.66 (m, 1H, H3′), 4.10 (m, 1H, H4′);(non-reducing end) 5.73 (bs, 1H, H1″), 4.17 (m, 1H, H2″), 4.38 (m, 1H,H3″), 4.22 (m, 1H, H4″); 3.95-3.82 (H5, maltotriose); spacer: 4.03, 4.02(2×s, 2H, OCH₂C(O)), 3.73-3.59 (m, 36H, OCH₂ TEG, HEG), 3.38 (t, 2H,CH₂NH₂), 3.27, 3.26 (2×s, 2H, SCH₂(O)), 2.75 (2×t, 2H, CH₂S); peptide:7.81-7.52 (m, 5H, H_(arom) BS), 7.79, 7.676, 7.42 7.41, (4×d, 4H,H_(arom) APA), 4.87, 4.66 (2×t, 1H, αCH APA, J_(αCH,βCH)=7.4 Hz), 4.07(m, 1H, αCH Lys), 3.37-3.73 (m, 8H, εCH₂ Lys, βCH₂ APA, CH₂Npiperidine), 1.96-1.46 (m, 12H, CH₂ piperidine, β/γ,δCH₂ Lys);

ES-MS: [M+3H]³⁺801.2, [M+2H]²⁺1200.8.

Partially Protected Pentasaccharide 30

Known pentasaccharide 29 (53 mg, 49 μmol) (R. C. Buijsman et al., Chem.Eur. J. 1996, 2, 12, p1572-1577) was dissolved in dimethylformamide(0.25 mL) and water (1 mL) and treated withN-(benzyloxycarbonyloxy)-succinimide (18 mg, 72 μmol) andN-ethylmorpholine (18.6 mL). After stirring for 15 min, TLC analysis(ethyl acetate/pyridine/acetic acid/water, 5/7/1.6/4, v/v/v/v) revealedthe reaction to be complete and the reaction mixture was directlyapplied onto a RP-18 column, which was eluted with water/methanol (90/10to 60/40). The appropriate fractions were pooled and concentrated to asmall volume and applied on a Dowex 50 WX4-H⁻ ion-exchange column inwater. The eluate was concentrated in vacuo to yield pure 30 (54 mg, 91%yield).

Sulfated Pentasaccharide 32

Compound 30 (54 mg, 45 μmol) was dissolved in dimethylformamide (1 mL).Triethylamine sulfurtrioxide complex (0.51 g, 5 equiv for each hydroxylgroup) was added and the mixture was stirred under a nitrogen atmosphereat 55° C. for 16 h. The mixture was subsequently cooled to 0° C. andaqueous sodium hydrogen carbonate was added (5 equiv for each eq. oftriethylamine sulfirtrioxide complex). The mixture was stirred for 1 h,concentrated to a small volume and applied onto a Sephadex G-25 column,which was eluted with 10% acetonitril in water. The appropriatefractions were pooled and concentrated to a small volume, which wassubsequently passed through a column of Dowex 50 WX4 (Na⁺ form) elutedwith water. The eluate was concentrated and redissolved in 0.2 Nhydrogen chloride (1 mL) and allowed to stand for 16 h at 4° C. Thereaction mixture was neutralized with 0.1 N sodium hydroxide anddesalted on a Sephadex G-25 column and eluted with 10% acetonitril inwater to afford homogeneous 31. Compound 31 was dissolved intert-butanol/water (6/1, v/v, 20 mL) containing a few drops of aceticacid. The solution was stirred under a continuous stream of hydrogen inthe presence of 10% Pd/C (100 mg). After 3 h the Pd/C catalyst wasremoved by filtration and the filtrate was concentrated in vacuo tofurnish pure 32 (60 mg, 60% yield).

Condensation Coupling of Pentasaccharide 32 with Peptide 16

Pentasaccharide 32 (15 mg, 6.5 μmol) was dissolved in 0.1 M NaH₂PO₄buffer (2 mL, pH 7.5 and to this solution was added sulfo-SIAB™ (16 mg,33 μmol). After stirring 3 h in the dark, HPLC analysis (monoQ anionexchange) revealed the reaction to be complete and the crude 34 waspurified on a Superdex 30 column (10% acetonitril in water). Theappropriate fractions were pooled and concentrated in vacuo at lowtemperature (25° C). To a solution of NAPAP analogue 16 (9 mg, 14 μmol)in a mixture of methanol (1 mL) and 0.1 M Na₂HPO₄ buffer (0.75 mL, pH7.0), degassed by passing through helium and by sonification before use,was added tributylphosphine (3.9 μL, 15 μmol). After stirring for 1 hunder an argon atmosphere, HPLC analysis (Lichrospher® RP-18 column)indicated a complete cleavage of the 2-pyridinesulfenyl group. Asolution of derivatized pentasaccharide 34 in dimethylformamide (0.25mL) and 0.1 M Na₂HPO₄ buffer (0.50 mL, pH 7.0) was added and the mixturewas stirred for 3 h. The crude product was applied onto a Sephadex G-50column, which was eluted with 10% acetonitril in water. The appropriatefractions were pooled, concentrated to a small volume and desalted on aSuperdex 30 column, which was eluted with 10% methanol in water.Concentration and lyophilization yielded conjugate III as a white solid(9 mg, 52% yield).

¹H NMR (D₂O, 600 MHz, HH-COSY): δ3.60, 3.53, 3.43 (3×s, 9H,CH₃O_(E,G,H)); ring D: 5.53 (m, 1H, H1), 4.15 (m, 1H, H2), 4.58 (m, 1H,H3), 3.56 (m, 1H, H4), 3.92 (m, 1H, H5), 4.26, 4.13 (2×m, 2H, H6, H6′);ring E: 4.70 (d, 1H, H1, J_(1,2)=8.1 Hz,), 4.21 (m, 1H, H2), 3.62 (m,1H, H3), 3.92 (m, 1H, H4), 3.74 (m, 1H, H5); ring F: 5.39 (d, 1H, H1,J_(1,2)=3.8 Hz), 4.22 (m, 1H, H2), 4.56 (m, 1H, H3), 3.83 (t, 1H, H4,J_(3,4)=J_(4,5)=9.8 Hz), 4.12 (m, 1H, H5); ring G: 5.15 (bs, 1H, H1),4.35 (m, 1H, H2), 3.76 (m, 1H, H3), 4.21 (m, 1H, H4), 4.80 (m, 1H, H5);ring H: 5.10 (d, 1H, H1, J_(1,2)=3.6 Hz), 4.31 (m, 1H, H2), 4.54 (m, 1H,H3), 4.21 (m, 1H, H4); spacer: 7.51, 7.53, 7.13, 712 (4×d, 4H, H_(arom)SIAB), 3.73 (m, 2H, CH₂CH₂NH₂), 3.66 (m, 12H, OCH₂ TEG), 3.31 (m, 2H,CH₂NH₂); peptide: 8.27, 8.22 (2×s, 1H, H_(arom) NAS), 7.98-7.60 (m, 6H,H_(arom) NAS), 7.71, 7.64, 7.46, 7.44 (4×d, 4H, H_(arom) APA), 4.60,4.45 (2×t, 1H, αCH APA, J_(αCH,βCH)=6.6 Hz), 4.00, 3.97 (2×m, 1H, αCHCys), 3.10-2.85 (m, 4H, CH₂N piperidine), 2.82-2.70 (m, 3H, βCH₂ Cys,βCH APA), 2.61 (mn, 1H, βCH′ APA), 1.55-1.15 (m, 6H, CH₂ piperidine);

ES-MS: [M−H]⁻2680.6

Using similar methods, the following compounds are prepared:

The biological activities of the compounds of the present invention weredetermined by the following test methods.

I. Anti-thrombin Assay

Thrombin (Factor IIa) is a factor in the coagulation cascade.

The anti-thrombin activity of compounds of the present invention wasassessed by measuring spectrophotometrically the rate of hydrolysis ofthe chromogenic substrate s-2238 exterted by thrombin. This assay foranti-thrombin activity in a buffer system was used to assess theIC₅₀-value of a test compound.

Test medium: Tromethamine-NaCl-polyethylene glycol 6000 (TNP) buffer

Reference compound: I2581 (Kabi)

Vehicle: TNP buffer.

Solubilisation can be assisted with dimethylsuiphoxide, methanol,ethanol, acetonitrfle or tert.-butyl alcohol which are without adverseeffects in concentrations up, to 2.5% in the final reaction mixture.

Technique Reagents*

1. Tromethamine-NaCl (TN) buffer

Composition of the buffer:

Tromethamine (Tris) 6.057 g (50 mmol) NaCl 5.844 g (100 mmol) Water to 1l

The pH of the solution is adjusted to 7.4 at 37° C. with HCl (10mmol·l⁻¹).

2. TNP buffer

Polyethylene glycol 6000 is dissolved in TN buffer to give aconcentration of 3 g·l⁻¹

3. S-2238 solution

One vial S-2238 (25 mg; Kabi Diagnostica, Sweden) is dissolved in 20 mlTN buffer to give a concentration of 1.25 mg·ml⁻¹ (2 mmol·l⁻¹).

4. Thrombin solution

Human thrombin (16 000 nKat·vial⁻¹; Centraal Laboratorium voorBloedtransfusie, Amsterdam, The Netherlands) is dissolved in TNP bufferto give a stock solution of 835 nKat·ml⁻¹.

Immediately before use this solution is diluted with TNP buffer to givea concentration of 3.34 nKat·ml⁻¹.

All ingredients used are of analytical grade

For aqueous solutions ultrapure water (Milli-Q quality) is used.

Preparation of test and reference compound solutions

The test and reference compounds are dissolved in Milli-Q water to givestock concentrations of 10⁻² mol·l⁻¹. Each concentration is stepwisediluted with the vehicle to give concentrations of 10⁻³, 10⁻⁴ and 10⁻⁵mol·l⁻¹. The dilutions, including the stock solution, are used in theassay (final concentrations in the reaction mixture: 3·10⁻³; 10⁻³;3·10⁻⁴; 10⁻⁴; 3·10⁻⁵; 10⁻⁵; 3·10⁻⁶ and 10⁻⁶ mol·l⁻¹, respectively).

Procedure

At room temperature 0.075 ml and 0.025 ml test compound or referencecompound solutions or vehicle are alternately pipetted into the wells ofa microtiter plate and these solutions are diluted with 0.115 ml and0.0165 ml TNP buffer, respectively. An aliquot of 0.030 ml S-2238solution is added to each well and the plate is pre-heated andpre-incubated with shaking in an incubator (Amersham) for 10 min. at 37°C. Following pre-incubation the hydrolysis of S-2238 is started byaddition of 0.030 ml thrombin solution to each well. The plate isincubated (with shaking for 30 s) at 37° C. Starting after 1 min ofincubation, the absorbance of each sample at 405 nm is measured every 2min. for a period of 90 min. using a kinetic microtiter plate reader(Twinreader plus, Flow Laboratories).

All data are collected in an IBM personal computer using LOTUS-MEASURE.For each compound concentration (expressed in mol·l⁻¹ reaction mixture)and for the blank the absorbance is plotted versus the reaction time inmin.

Evaluation of responses: For each final concentration the maximumabsorbance was calculated from the assay plot. The IC₅₀-value (finalconcentration, expressed in μmol·l⁻¹, causing 50% inhibition of themaximum absorbance of the blank) was calculated using the logittransformation analysis according to Hafner et al.(Arzneim.-Forsch./Drug Res. 1977; 27(II): 1871-3).

Antithrombin Activity

Example IC₅₀ (mol · 1⁻¹) I (one diastereomer) 2 × 10⁻⁷ II 8 × 10⁻⁶ III3.5 × 10⁻⁷  

II. Anti-factor Xa Assay

Activated Factor X (Xa) is a factor in the coagulation cascade. Theanti-Xa activity of compounds of the present invention was assessed bymeasuring spectrophotometrically the rate of hydrolysis of thechromogenic substrate s-2222 exterted by Xa. This assay for anti-Xaactivity in a buffer system was used to assess the IC₅₀-value of thetest compound.

In general the followed procedure and test conditions were analogous tothose of the anti-thrombin assay as described above. Differences areindicated below.

Reference compound: benzamidine

Vehicle: TNP buffer.

Solubilisation can be assisted with dimethylsulphoxide, methanolethanol, acetonitrile or tert.-butyl alcohol which are without adverseeffects in concentrations up to 1% (for DMSO) and 2.5% (for the othersolvents) in the final reaction mixture.

Technique Reagents*

3. S-2222 solution

One vial S-2222 (15 mg; Kabi Diagnostica, Sweden) is dissolved in 10 mlwater to give a concentration of 1.5 mg·ml⁻¹ (2 mmol·l⁻¹).

4. Xa solution

Bovine Factor Xa Human (71 nKat·vial⁻¹; Kabi Diagnostica) is dissolvedin 10 ml TNP buffer and then further diluted with 30 ml TNP buffer togive a concentration of 1.77 nKat·ml⁻¹. The dilution has to be freshlyprepared.

Procedure

Instead of the S-2238 solution (in anti-thrombin assay), the aboveS-2222 solution is added to each well in this assay.

Anti-factor Xa activity

Example U/mg III 885

What is claimed is:
 1. A compound of the formula (I)

wherein R¹ is phenyl, naphthyl, 1,2,3,4-tetrahydronaphthyl,(iso)quinolinyl, tetrahydro(iso)quinolinyl,3,4-dihydro-1H-isoquinolinyl, chromanyl or the camphor group, whichgroups may optionally be substituted with one or more substituentsselected from (1-8C)alkyl or (1-8C)alkoxy; R² and R³ are independently Hor (1-8C)alkyl; R⁴ is (1-8C)alkyl or (3-8C)cycloalkyl; or R³ and R⁴together with the nitrogen atom to which they are bonded are anonaromatic (4-8)membered ring optionally containing another heteroatom,the ring optionally being substituted with (1-8C)alkyl orSO₂-(1-8C)alkyl; Q is a spacer having a chain length of 10 to 70 atoms;and Z is a negatively charged oligosaccharide residue comprising two tosix monosaccharide units, the charge being compensated by positivelycharged counterions; or a pharmaceutically acceptable salt thereof or aprodrug thereof.
 2. The compound of claim 1, wherein Z is derived froman oligosaccharide which has AT-III mediated anti-Xa activity.
 3. Thecompound of claim 2, wherein Z is a pentasaccharide residue.
 4. Thecompound of claim 3, wherein Z has the formula (II)

wherein R⁵ is independently OSO₃ ⁻¹ or (1-8C)alkoxy.
 5. The compound ofclaim 1, wherein R¹ is is phenyl, 4-methoxy-2,3,6-trimethylphenyl ornaphthyl; R² is H; and NR³R⁴ represents the piperidinyl group.
 6. Thecompound of claim 1, wherein Q has the formula (III)—[(CH₂)₂O]_(m)—[(CH₂)_(n)—NR³—C(O)]_(p)—W—(CH₂)_(s)—  (III), wherein Wis —[1,4-phenylene-NR³—C(O)]_(q)—(CH₂)_(r)—S—or—(CH₂)_(t)—S—(CH₂)_(u)—[O(CH₂)₂]_(v)—O—(CH₂)_(w)—C(O)—NR³—; and R³ isindependently H or (1-8C)alkyl; m=1-12; n=1-8; p=0-4; q=0 or 1; r=1-8;s=1-8; t=1-8; u=1-8; v=1-12; w=1-8; the total number of atoms is 10-70;and the moiety —[(CH₂)₂O]_(m)— is the end with which Q is attached to Z.7. The compound of claim 6, wherein Q is selected from—[(CH₂)₂O]₅—(CH₂)₂—NH—C(O)—CH₂—S—CH₂—;—[(CH₂)₂O]₅—(CH₂)₂—NH—C(O)—CH₂—S—(CH₂)₂—[O(CH₂)₂]₃—O—CH₂—C(O)—NH—(CH₂)₄—;and —[(CH₂)₂O]₃—(CH₂)₂—NH—C(O)-1,4-phenylene-NH—C(O)—CH₂—S—CH₂—.
 8. Apharmaceutical composition comprising the compound of claim 1 and apharmaceutically acceptable auxiliary.
 9. A method of anti-thrombintherapy comprising administering an effective amount of a compoundaccording to claim 1.